Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne.

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients.

Whole body vibration therapy in patients with Duchenne muscular dystrophy--a prospective observational study.

OBJECTIVES: To study the tolerability of whole body vibration (WBV) exercise in patients with Duchenne muscular dystrophy (DMD) and its effects on muscle and bone. METHODS: WBV was performed two to three times a week for three months. Motor function, muscle strength, bone mass and biochemical markers of bone and mineral metabolism were analyzed before and after the WBV period at 0, 3, 6 and 12 months. RESULTS: Six ambulatory patients with DMD aged 5.7-12.5 years completed the study. No changes in creatine kinase activity were found, indicating that the WBV exercise did not further damage the skeletal muscle. No significant changes in bone mass, muscle strength or bone markers were found. However, there was a non-significant trend for the bone formation marker, bone-specific alkaline phosphate, to increase from a mean of 59 U/L to 73 U/L after three months of WBV. The bone formation marker levels returned to baseline three months after discontinuing WBV and were still at that level after nine months. CONCLUSIONS: WBV therapy appears to be safe and well tolerated among ambulatory DMD patients. The potential benefits of WBV on bone and muscle in DMD remain to be elucidated.

Ophthalmological findings in children and young adults with genetically verified mitochondrial disease.

AIM: To describe ophthalmological phenotypes in patients with mitochondrial disease and known genotypes. METHODS: A retrospective study was performed on 59 patients (29 male, 30 female) with a mean age of 11.8 years who had mitochondrial disease with known DNA mutations. Fifty-seven of the 59 subjects underwent a detailed ophthalmological examination including visual acuity (VA), eye motility, refraction, slit-lamp examination, ophthalmoscopy and, in almost one-half of the cases, a full-field electroretinogram (ERG). RESULTS: Forty-six (81%) of the patients had one or more ophthalmological findings such as ptosis (n = 16), reduced eye motility (n = 22) including severe external ophthalmoplegia (n = 9), strabismus (n = 4), nystagmus (n = 9), low VA (n = 21), refractive errors (n = 26), photophobia (n = 4), and partial or total optic atrophy (n = 25). Pigmentation in the macula and/or periphery was noted in 16 patients. In 10/27 investigated individuals with full field ERG, retinal dystrophy was recorded in six different genotypes representing Kearns-Sayre syndrome (n = 5), Leigh syndrome (n = 1), Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (n = 1), Myoclonus epilepsy with red ragged fibres (MERRF) (n = 1), Leber hereditary optic neuropathy (n = 1) and mitochondrial myopathy (n = 1). CONCLUSION: The results show that a majority of patients with mitochondrial disorders have ophthalmological abnormalities. We recommend that an ophthalmological examination, including ERG, be performed on all children and adolescents who are suspected of having a mitochondrial disease.

Progressive encephalopathy and complex I deficiency associated with mutations in MTND1.

Complex I of the oxidative phosphorylation system is composed of at least 45 subunits, seven of which are encoded by mitochondrial DNA (mtDNA). In this study we have investigated two children with complex I deficiency in muscle mitochondria. Patient 1 had cerebellar ataxia from early infancy and an abnormal MRI of the brain compatible with Leigh syndrome (LS). The course was rapidly progressive with frequent exacerbations and death at 2 years and 10 months of age. Patient 2 had a lactic acidosis in the newborn period and had a severe psychomotor developmental retardation. In her teens she developed hypertrophic cardiomyopathy and died at 26 years of age because of cardiac insufficiency. Sequencing analysis of mitochondrial encoded ND genes (MTND) showed two DE NOVO mutations in MTND1 in both patients. Patient 1 had a novel heteroplasmic G3890A mutation, R195Q. Patient 2 had a heteroplasmic G3481A mutation, E59K. The G3890A mutation in patient 1 is the first identified mutation in MTND1 in association with LS and complex I deficiency. The findings in this patient as well as in patient 2 demonstrate new clinical expressions of mutations in MTND1. The findings in patient 2 also illustrates that MTND mutations may be pathogenic even at a low percentage.

Inflammation and response to steroid treatment in limb-girdle muscular dystrophy 2I.

Limb-girdle muscular dystrophy (LGMD) type 2I, caused by mutations in the fukutin-related protein gene (FKRP), is one of the most common forms of LGMD in childhood. We describe two patients with LGMD2I and a Duchenne-like phenotype. In addition to the common L276I mutation, both patients had a new mutation in FKRP, L169P and P89L, respectively. Clinical onset was triggered by viral upper respiratory tract infections. In addition to the common dystrophic pattern with a weak immune histochemical staining for alpha-dystroglycan, muscle biopsy showed inflammatory changes. This was especially striking in one of the patients with up-regulation of MHC class 1 antigen, suggestive of myositis. Both patients showed a good clinical response to treatment with prednisolone, which was initiated at daily dosage of 0.35 mg/kg/day. Our results provide evidence for an inflammatory involvement in the pathological expression of LGMD2I and open up the possibility that this disorder could be treatable with corticosteroids.

Distal arthrogryposis and muscle weakness associated with a beta-tropomyosin mutation.

Tropomyosin (TM), a sarcomeric thin-filament protein, plays an essential part in muscle contraction by regulating actin-myosin interaction. We describe two patients, a woman and her daughter, with muscle weakness and distal arthrogryposis (DA) type 2B, caused by a heterozygous missense mutation, R133W, in TPM2, the gene encoding beta-TM. Our results demonstrate the involvement of muscle dysfunction in the pathogenesis of DA and the fact that DA2B may be caused by mutations in TPM2.

A mutation in the fast skeletal muscle troponin I gene causes myopathy and distal arthrogryposis.

OBJECTIVE: To describe a three-generation family with distal arthrogryposis associated with myopathy and caused by a mutation in the gene encoding for sarcomeric thin filament protein troponin I, TNNI2. METHODS: The authors performed clinical investigations and reviewed medical records. Muscle biopsy specimens were obtained for morphologic analysis. Genomic DNA was extracted from blood and analyzed for mutations in TNNI2. RESULTS: The five affected individuals had predominantly distal congenital joint contractures, mild facial involvement (mild micrognathia, narrow palpebral fissures), and no detectable muscle weakness. The four affected adults had slightly increased levels of creatine kinase in blood, and muscle biopsy specimens showed findings of myopathy with changes restricted to type 2 fibers. These included variability of muscle fiber size, internalized nuclei, and increased interstitial connective tissue. Analysis of TNNI2 encoding the troponin I isoform expressed in type 2 muscle fibers disclosed a heterozygous three-base in-frame deletion, 2,918-2,920del, skipping the highly conserved lysine at position 176. The mutation was present in all 5 affected individuals but was not identified in any of the 11 unaffected family members. CONCLUSION: Distal arthrogryposis type 1 is genetically heterogeneous, and myopathy due to sarcomeric protein dysfunction may be one underlying cause of the disease.

Mitochondrial myopathy with exercise intolerance and retinal dystrophy in a sporadic patient with a G583A mutation in the mt tRNA(phe) gene.

We describe a second patient with the 583G>A mutation in the tRNA(phe) gene of mitochondrial DNA (mtDNA). This 17-year-old girl had a mitochondrial myopathy with exercise intolerance and an asymptomatic retinopathy. Muscle investigations showed occasional ragged red fibers, 30% cytochrome c oxidase (COX)-negative fibers, and reduced activities of complex I+IV in the respiratory chain. The mutation was heteroplasmic (79%) in muscle but undetectable in other tissues. Analysis of single muscle fibers revealed a significantly higher level of mutated mtDNA in COX-negative fibers. Our study indicates that the 583G>A mutation is pathogenic and expands the clinical spectrum of this mutation.

Two new mutations in the MTATP6 gene associated with Leigh syndrome.

In this study we have analyzed the mtDNA encoded ATPase 6 and 8 genes ( MTATP6 and MTATP8) in two children with Leigh syndrome (LS) and reduced Mg (2+) ATPase activity in muscle mitochondria. In patient 1, with a mild and reversible phenotype, mutational analysis revealed a heteroplasmic T --> C mutation at nt position 9185 (T9185C) in the MTATP6. The mutation resulted in substitution of a highly conserved leucine to proline at codon 220. The proportion of the mutation was > 97 % in the patient's blood and muscle and 85 % in blood of his asymptomatic mother. Patient 2, with severe clinical phenotype and death at 2 years of age, exhibited a novel heteroplasmic T9191C missense mutation in the MTATP6, which converted a highly conserved leucine to a proline at position 222 of the polypeptide. The proportion of the mutation was 90 % in fibroblasts and 94 % muscle tissue. This mutation was absent in the patient's parents and sister suggesting that the mutation was de novo. Our findings expand the spectrum of mutations causing LS and emphasize the role of MTATP6 gene mutations in pathogenesis of LS.

Genotypes and clinical phenotypes in children with cytochrome-c oxidase deficiency.

Cytochrome c oxidase (COX) deficiency has been associated with a wide spectrum of clinical features and may be caused by mutations in different genes of both the mitochondrial and the nuclear DNA. In an attempt to correlate the clinical phenotype with the genotype in 16 childhood cases, mtDNA was analysed for deletion, depletion, and mutations in the three genes encoding COX subunits and the 22 tRNA genes. Furthermore, nuclear DNA was analysed for mutations in the SURF1, SCO2, COX10, and COX17 genes and cases with mtDNA depletion were analysed for mutations in the TK2 gene. SURF1-mutations were identified in three out of four cases with Leigh syndrome while a mutation in the mitochondrial tRNA (trp) gene was identified in the fourth. One case with mtDNA depletion had mutations in the TK2 gene. In two cases with leukoencephalopathy, one case with encephalopathy, five cases with fatal infantile myopathy and cardiomyopathy, two cases with benign infantile myopathy, and one case with mtDNA depletion, no mutations were identified. We conclude that COX deficiency in childhood should be suspected in a wide range of clinical settings and although an increasing number of genetic defects have been identified, the underlying mutations remain unclear in the majority of the cases.

SURF1 gene mutations in three cases with Leigh syndrome and cytochrome c oxidase deficiency.

Leigh syndrome (LS) is one of the most frequent forms of mitochondrial disease in infancy and childhood. Mutations in SURF1 have been shown to be an important cause of LS with cytochrome c oxidase (COX) deficiency. The authors have identified four pathogenic mutations including a novel, in-frame, 15-bp tandem duplication (806-820) in exon 8 and a novel 751+1G>A splice site mutation in SURF1 in three cases of LS with COX deficiency.

Leigh syndrome with cytochrome-c oxidase deficiency and a single T insertion nt 5537 in the mitochondrial tRNATrp gene.

We report a nine-year-old boy with the features of Leigh syndrome (LS) and a severe cytochrome-c oxidase (COX) deficiency with a single thymidine insertion at nucleotide position 5537 (T 5537i) in the tRNA Trp gene of mitochondrial DNA. During infancy the boy was irritable and hypotonus was noticed. Early motor development was delayed, although mental development seemed normal until eight months of age. Early neurological signs were nystagmus, hypertonus and optic atrophy. Severe seizures and mental retardation developed subsequently. Major findings on neuroradiological investigation were from the brainstem, thalami and white matter compatible with LS. Spectrophotometric analysis of skeletal muscle mitochondria showed a profound COX deficiency and a marked complex I deficiency. Enzyme-histochemical analysis showed reduced COX activity in the majority of the muscle fibres. There were no ragged red fibres. The T 5537i mutation was found in a high proportion (> 95 %) in blood, liver and muscle tissue of the patient and in blood of the patient's mother (81 %). This mutation has previously been described in one family in which one child had a very high proportion of the T 5537i mutation and clinical features of LS. We conclude that, although mtDNA mutations are considered to be rare in LS with COX deficiency, the T 5537i mutation should be screened for in cases of LS with COX deficiency when SURF1 gene mutations have been excluded, especially when complex I activity is also decreased.

Cardiomyopathy in children with mitochondrial disease; clinical course and cardiological findings.

AIMS: To determine the frequency of cardiomyopathy in children with mitochondrial disease and describe their clinical course, prognosis and cardiological manifestations. METHODS AND RESULTS: Of 301 children with CNS and neuromuscular disease referred to our institution in 1984 to 1999, 101 had mitochondrial disease. Seventeen patients had cardiomyopathy, diagnosed by echo-Doppler investigations, all of the hypertrophic, non-obstructive type. The onset of symptomatic mitochondrial disease ranged from birth to 10 years of age. Eight children had cytochrome-c oxidase deficiency, while the remaining nine had various defects. Cardiomyopathy was diagnosed from birth to 27 years. Left ventricular posterior wall and septal thickness were both increased: z-scores +4.6+/-2.6 and +4.3+/-1.6 (mean+/-SD), respectively. The left ventricular diastolic diameter z-score, +1.3+/-3.4, and fractional shortening, 24+/-13%, displayed marked variations. Nine patients developed heart failure. Eleven patients with cardiomyopathy died, including all eight with cytochrome-c oxidase deficiency, and one patient underwent a heart transplantation. Mortality in children with mitochondrial disease was higher in those with cardiomyopathy (71%) than those without (26%) (P<0.001). CONCLUSIONS: In children with mitochondrial disease, cardiomyopathy was common (17%) and was associated with increased mortality. The prognosis for children with cytochrome-c oxidase deficiency and cardiomyopathy appeared to be particularly unfavorable.

Muscle strength and motor function in children and adolescents with spinal muscular atrophy II and III.

The aim of this study was to quantify isometric muscle strength and motor function in children and adolescents with spinal muscular atrophy (SMA) and to analyse the impact of reduced muscle strength on motor function. Six children and adolescents with SMA II and eight with SMA IlI were assessed regarding isometric muscle strength and motor function. Isometric muscle strength was tested with a myometer and the values obtained were compared with normative data. Motor function was videotaped and 20 movements were scored according to a three-point scale. All of the assessed children and adolescents with SMA II and SMA III showed reduced muscle strength, but there were great differences within the group. The typical pattern of muscle weakness in SMA, with proximal weakness greater than distal and the lower limbs more affected than the upper, was also seen in these children. The muscle weakness affected motor function in all assessed children. Walking, transfer from lying or sitting to the standing position and stair-climbing were possible in some of the children, despite marked reduction of muscle strength. The study increases our knowledge concerning the degree of muscle weakness in children with SMA and the impact of muscle weakness on motor function. The results increase our possibilities of understanding the prerequisites for everyday life in these children and planning therapeutic interventions. Repeated assessments with the methods used in this study may be used to monitor the course of the disease and to evaluate the efficacy of treatment.

The incidence of mitochondrial encephalomyopathies in childhood: clinical features and morphological, biochemical, and DNA abnormalities.

In this study we present incidence, point prevalence, and mortality figures of mitochondrial encephalomyopathies in a population-based study of children from western Sweden. Through the screening of registers and review of medical records, we identified 32 patients under 16 years of age from the study population who were diagnosed between January 1, 1984, and December 31, 1998. The incidence of mitochondrial encephalomyopathies in preschool children (<6 years of age) was 1 out of 11,000. The preschool incidence of Leigh's syndrome was 1 out of 32,000, and the preschool incidences of both Alper's syndrome and infantile mitochondrial myopathy with cytochrome C oxidase deficiency were 1 out of 51,000. The point prevalence January 1, 1999) of mitochondrial encephalomyopathies in children under 16 years of age was 1 out of 21,000. The median survival for patients with infantile onset was until 12 years of age. We identified 4 cases with mitochondrial DNA point mutations, 2 cases with mitochondrial DNA deletions, and 2 cases with nuclear mutations in the SURF1 gene. We conclude that mitochondrial encephalomyopathies are relatively common neurometabolic disorders in childhood.

Neuromuscular disorders in childhood: a descriptive epidemiological study from western Sweden.

A retrospective epidemiological study of neuromuscular disorders was carried out in children born between 1979 and 1994 in western Sweden. The purpose was to determine overall and specific prevalences, overall cumulative incidence and birth incidences of selected disorders. Cases were ascertained from 12 different sources and medical records, investigations and diagnosis were reviewed. We found a point prevalence in the population < 16 years of age of 63.1 x 10(-5) for all neuromuscular disorders and 53.1 x 10(-5) for inherited neuromuscular disorders. The point prevalence in children of school age was even higher. We found a higher occurrence of hereditary motor and sensory neuropathy, congenital myopathies and mitochondrial encephalo-myopathy, a slightly lower occurrence of Duchenne muscular dystrophy and spinal muscular atrophy and equal occurrence of myotonic dystrophy compared to previous studies in other countries. We conclude that neuromuscular disorders are more common in childhood than has previously been reported.

Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295-298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G-->A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered beta-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.

Threshold expression of the tRNA(Lys) A8344G mutation in single muscle fibres.

We investigated the distribution in skeletal muscle of mitochondrial DNA (mtDNA) with the tRNA(Lys) A8344G mutation, which is associated with myoclonus epilepsy and ragged red fibres (MERRF) syndrome. Isolated muscle fibre segments (n = 144) from six individuals of two different families carrying the mutation were studied. Two of these individuals were affected by MERRF while four had no or minor clinical symptoms. In one individual with a low overall level of mutated mtDNA (mean 18%) the variation in the proportion of mutated mtDNA between individual muscle fibres ranged from 0 to 80%. This result demonstrates that segregation of the tRNA(Lys) A8344G mutation within a tissue may lead to very marked variation of the level of mutated mtDNA between individual cells. There was a very high apparent threshold level of mutated mtDNA (95.3-97.7%) for expression of histochemical cytochrome c oxidase (COX) deficiency in individual muscle fibres. The results indicated that this apparent threshold level varied slightly between patients. Ultrastructural examination revealed that an appreciable proportion of the mitochondria in COX-positive muscle fibres lacked COX activity. Variation in intercellular and interorganellar distribution of mutated mtDNA in addition to the absolute mtDNA copy number may explain differences in clinical phenotypes in patients with high levels of the tRNA(Lys) A8344G mutation.